1. Field
The disclosed subject matter generally relates to phase contrast imaging, e.g., phase contrast microscopy.
2. Discussion of Related Art
Translucent objects or phase objects can alter only the phase of the optical wave, not its amplitude. Hence, these objects are very difficult to see with the naked eye and cannot be captured by an ordinary camera. A phase contrast microscope can be used to obtain high-contrast images of transparent specimens, such as living cells (usually in culture), micro-organisms, thin tissue slices, lithographic patterns, fibers, latex dispersions, glass fragments, and subcellular particles (including nuclei and other organelles). One useful feature of a phase contrast microscope is that living cells can be examined in their natural state without being fixed, and/or stained. As a result, the dynamics of ongoing biological processes can be observed and recorded in high contrast with sharp clarity of minute specimen details.
In 1933, Zernike developed a non-destructive mechanism based on the principle of phase contrast to observe translucent microscopic objects. It is a two step process: (1) separation of deviated and undeviated components in the light transmitted through the specimen with a π/2 phase difference between them and (2) obtaining an additional π/2 phase separation thereby converting phase information into amplitude (intensity) contrast for display. If the undeviated light is phase shifted by π/2, then the undeviated and diffracted light arriving at the eyepiece would produce destructive interference and the object details appear dark in lighter background. This is known as dark or positive phase contrast. If, however, the undeviated light is phase shifted by −π/2 then the diffracted and undeviated light beams interfere constructively. This produces a bright image of the details of the specimen in dark background and is known as negative or bright contrast. This principle is exploited for the phase contrast microscope.
Existing phase contrast microscopes employ a tungsten-halogen lamp as a light source and a condenser annulus for separation of the deviated and undeviated light. They also use phase plates for generating the additional phase retardation between undeviated light and light diffracted by the object, thereby transforming minute variations in phase of the object into corresponding changes in image contrast. The collimated light passes through the condenser plate which typically contains several transparent annular rings (carefully positioned and designed to be an optical conjugate to a phase plate residing in the image plane) and is focused onto the specimen. The light transmitted by the specimen consists of undeviated light and diffracted light. The undeviated and diffracted light differs in phase by π/2 due to the inherent phase variations in the specimen. The light is then collected by the objective and is spatially separated at its back focal plane. A phase plate selectively placed at this back focal plane introduces an additional π/2 relative phase difference. Thus the undeviated and diffracted light interferes destructively so that the phase variations in the specimen appear bright against a dark background. Two types of phase plates, positive and negative, are available to produce a bright image in dark background or vice versa.
However, there are some unavoidable disadvantages associated with the use of these plates:
1. Halo and shade-off contrast patterns are frequently observed in phase contrast images. These observed intensity patterns do not directly correspond to the optical path difference between the specimen and the surrounding medium. The artifacts depend on both the geometrical and optical properties of the phase plate and the specimen being examined. In particular, the width and transmittance of the phase plate material play a critical role in controlling these effects. In addition, these effects are heavily influenced by the objective magnification. Apodized phase plates are used for reducing the severity of halos.
2. In order to resolve minute details and edges in the specimen, a large angle of diffracted light must be captured by the microscope objective and must be brought into a sharp focus at the image plane. The condenser aperture diaphragm opening size partially controls the coherence of the light incident on the specimen. Decreasing the opening size of the diaphragm yields greater spatial coherence but it introduces diffraction related artifacts. Thus the system is limited by the working numerical aperture of the objective thereby reducing the resolution of the instrument.
3. When the object is changed or a different magnification is desired, the bright-field image has to be obtained first and then the condenser plate has to be rotated to position the annular ring to match the new phase plate. Thus as a result of frequent rotations of the condenser plate, the annular ring tends to be out of alignment with the phase plate requiring regular maintenance of the system. Special tools are provided for adjusting the condenser plate, which require skill and experience on the part of the operator. Furthermore, rotation of the condenser plate can sometimes cause the specimen to move as it is positioned just before the condenser.
Existing phase contrast microscopes have been modified since their invention, in terms of phase plate design and detection schemes. However, conventional phase contrast microscopes do not exploit advantages that come with a coherent source. For example, the white light sources of conventional phase contrast microscopes cannot provide Fourier transformation, as a result which the object information cannot be well separated at the Fourier plane.
With the growing demand for a variety of imaging modalities that offer different distinct advantages, improved methods for imaging phase objects in transparent media and imaging phase objects in tissue-like scattering media are needed.